Skeletal muscle from TBC1D4 p.Arg684Ter variant carriers is severely insulin resistant but exhibits normal metabolic responses during exercise

Abstract

In the Greenlandic Inuit population, 4% are homozygous carriers of a genetic nonsense TBC1D4 p.Arg684Ter variant leading to loss of the muscle-specific isoform of TBC1D4 and an approximately tenfold increased risk of type 2 diabetes¹. Here we show the metabolic consequences of this variant in four female and four male homozygous carriers and matched controls. An extended glucose tolerance test reveals prolonged hyperglycaemia followed by reactive hypoglycaemia in the carriers. Whole-body glucose disposal is impaired during euglycaemic-hyperinsulinaemic clamp conditions and associates with severe insulin resistance in skeletal muscle only. Notably, a marked reduction in muscle glucose transporter GLUT4 and associated proteins is observed. While metabolic regulation during exercise remains normal, the insulin-sensitizing effect of a single exercise bout is compromised. Thus, loss of the muscle-specific isoform of TBC1D4 causes severe skeletal muscle insulin resistance without baseline hyperinsulinaemia. However, physical activity can ameliorate this condition. These observations offer avenues for personalized interventions and targeted preventive strategies.

Fig. 1. Homozygous TBC1D4 p.Arg684Ter variant carriers display prolonged hyperglycaemia followed by hypoglycaemia during an extended oral glucose challenge.

a, Graphical representation of the OGTT. b–i, Blood glucose (b) as well as plasma C-peptide (c), insulin (d), glucagon (e), adrenaline (f), noradrenaline (g), cortisol (h) and growth hormone (i) during an extended (6 h) OGTT. *Difference between TBC1D4 carriers and Controls at the given time point. ‡Different from basal (time 0) within the same group. One symbol P < 0.05. Two symbols P < 0.01. Three symbols P < 0.001. Symbols within parentheses represent P values between 0.1 and 0.05. n = 8 in Controls and n = 7 in TBC1D4 carriers. Data are means ± s.e.m. Data were analysed using a two-way repeated ANOVA test (one factor repeated) and two-tailed Student–Newman–Keuls post hoc analyses for multiple comparisons. Graphics in a created using BioRender.com.

Fig. 2. Compromised peripheral glucose disposal during EHC conditions but normal liver and adipose tissue regulation in homozygous TBC1D4 p.Arg684Ter variant carriers.

a, Graphical representation of the EHC. b, GIR during the insulin clamp. c–e, GIR (c), endogenous glucose rate of appearance (RA) (d) and glucose rate of disappearance (RD) (e) at 120 min of the clamp. f, Plasma FAs during the clamp. g, Average leg glucose uptake during the last 40 min of the clamp. h, Targeted immunoblotting analyses of proteins associated with glucose metabolism, fat metabolism and the mitochondrial electron transport chain in skeletal muscle. i, Volcano plot showing proteome log₂ fold change (FC) (carriers/controls) plotted against –log₁₀ P value highlighting downregulated proteins TBC1D4, TBC1D1 and GLUT4 (SLC2A4). *Difference between TBC1D4 carriers and controls at the given time point. ‡Different from basal (time 0) within the same group. n = 5 (b–g, i). n = 7 (h) except for mTOR, p70S6K and ACC (n = 5 in controls and TBC1D4 carriers) as well as Akt2, AMPKα2, CS, CD36 and FATP4 (n = 6) in controls and GLUT1 (n = 6) in TBC1D4 carriers. Data are means ± s.e.m. Data were analysed using a two-tailed paired Student’s t-test (c–e, g), a two-tailed non-paired Student’s t-test (h) as well as a two-way repeated ANOVA test (two-factor repeated) and two-tailed Student–Newman–Keuls post hoc analyses for multiple comparisons (b, f). Graphics in a created using BioRender.com. a.u., arbitrary units.

Fig. 3. Improvement of skeletal muscle insulin resistance by a single bout of exercise in homozygous TBC1D4 p.Arg684Ter variant carriers.

a, Graphical representation of the EHC 3 h into recovery from one-legged knee-extensor exercise. b–g, Phosphorylation of Akt Thr308 (b), p70S6K Thr389 (c), TBC1D4 Thr642 (e), TBC1D1 Thr596 (f) and TBC1D4 Ser704 (g) as well as GS activity (d) in rested and previously exercised muscle before and at the end of the insulin clamp. h, Representative immunoblots. i, Average glucose uptake in the rested and exercised leg during the last 40 min of the insulin clamp. j, Delta leg glucose uptake (glucose uptake in exercised leg minus glucose uptake in a rested leg). *Difference (main effect) between TBC1D4 carriers and controls. ‡Difference (main effect) between rested and exercised leg. n = 5 (rested and exercised leg − insulin), n = 3 (rested leg + insulin) and n = 4 (exercised leg + insulin) in controls and n = 5 in TBC1D4 carriers except for GS activity with n = 4 (rested leg − insulin) in controls and TBC1D4 carriers (b–g). The difference in n is due to missing biopsies as well as depleted sample material. n = 5 in controls and in TBC1D4 carriers (i,j). Data are means ± s.e.m. Data were analysed using a two-tailed paired Student’s t-test (j) as well as a two-way repeated ANOVA test (two-factor repeated) and two-tailed Student–Newman–Keuls post hoc analyses for multiple comparisons (b–g, i). Graphics in a created using BioRender.com. a.u., arbitrary units. Source data

Fig. 4. Intact skeletal muscle glucose uptake and cellular signalling during exercise in homozygous TBC1D4 p.Arg684Ter variant carriers.

a, Graphical representation of the one-legged knee-extensor exercise model. b, Leg glucose uptake during 1 h of knee-extensor exercise and 3 h recovery. Leg glucose uptake in a rested leg before exercise: 6.7 ± 3.5 (controls) and 6.4 ± 1.8 (carriers) μmol min⁻¹ kg⁻¹ LLM. Average leg glucose uptake in a rested leg in exercise recovery: 4.3 ± 1.4 (controls) and 5.5 ± 1.4 (carriers) μmol min⁻¹ kg⁻¹ LLM. c,d, Skeletal muscle glycogen content (c) and GS activity (d) in a rested leg (rest) and exercised leg (exercise). e–l, Skeletal muscle protein phosphorylation of GS site 2 + 2a (Ser7 + Ser10) (e), AMPKα Thr172 (f), ACC Ser221 (g), P38 Thr180/Tyr182 (h), TBC1D1 Ser237 (i), PDH site 1 (Ser293) (j), p70S6K Thr389 (k) and TBC1D4 Ser704 (l). m, Representative immunoblots. ‡Different from basal (time 0) and rest in both groups (main effects). n = 5 in controls and in TBC1D4 carriers. Data are means ± s.e.m. Data were analysed using a two-way repeated ANOVA test (two-factor repeated) and two-tailed Student–Newman–Keuls post hoc analyses for multiple comparisons. Graphics in a created using BioRender.com. a.u., arbitrary units. Source data

Extended Data Fig. 1. Gut hormones during an extended oral glucose challenge in homozygous TBC1D4 p.Arg684Ter variant carriers.

a, b, Plasma levels of GIP (a) and GLP-1 (b) during an extended (6-h) oral glucose tolerance test (OGTT). ‡Different from basal (time = 0) in both groups. Three symbols = P < 0.001. n = 8 in Controls and n = 7 in TBC1D4 carriers. Data are means ± SEM. Data were analyzed using a two-way repeated ANOVA test (one factor repeated) and two-tailed Student–Newman-Keuls post hoc analyses for multiple comparisons.

Extended Data Fig. 2. Glycemic markers, oxidative and non-oxidative glucose disposal as well as adipose tissue GSEA during euglycemic-hyperinsulinemic clamp conditions in homozygous TBC1D4 p.Arg684Ter variant carriers.

a, Basal (Pre-clamp) endogenous glucose rate of appearance (Ra). b, c, Blood glucose levels (b) and plasma insulin concentrations (c) during the insulin clamp. d, Plasma fatty acids during an extended (6-h) oral glucose tolerance test (OGTT). e, RNA Gene Set Enrichment Analysis (GSEA) in adipose tissue obtained at the end of the insulin clamp with number of genes included in the gene set indicated by ‘Size’, Enrichment Score indicated by ‘ES’, Normalized Enrichment Score indicated by ‘NES’, Nominal P Value indicated by ‘NOM p-value’ and False Discovery Rate indicated by ‘FDR q-value’. f, Leg glucose uptake during the 120 min insulin clamp. g, Glucose disappearance rate displayed as oxidative and non-oxidative glucose metabolism during the insulin clamp steady state period (90 min). ‡Different from basal (time = 0) in both groups (line indicates main effect). *Difference between TBC1D4 carriers and Controls. One symbol = P < 0.05, two symbols = P < 0.01 and three symbols = P < 0.001. n = 5 in both groups (a–c, f, g) and n = 8 in Controls and n = 7 in TBC1D4 carriers (d). Data are means ± SEM. Data were analyzed using a two-tailed paired students t-test (a, g) as well as a two-way repeated ANOVA test (one factor repeated (d) and two factor repeated (b, c)) and two-tailed Student-Newman-Keuls post hoc analyses for multiple comparisons (b–d). Statistical analyses were not applied to data in panel f. LLM, leg lean mass. AU, arbitrary units.

Extended Data Fig. 3. Muscle fiber type-specific protein content and GLUT4 immunostaining in muscle fibers from homozygous TBC1D4 p.Arg684Ter variant carriers.

a, Representative immunoblots for data related to figure panel 2 h. b, c, Distribution of Myosin Heavy Chains (MHC) (b) and regulatory metabolic (c) proteins in MHC-defined type 1 and type 2 skeletal muscle fiber bundles. d, Representative immunoblots. e, Quantification of GLUT4 imaging by confocal microscopy of isolated skeletal muscle fibers. n = 7 in Controls and n = 6 in TBC1D4 carriers (b, c). n = 15 fibers obtained from 3 subjects (4 to 6 fibers/subject) in each group (e). Data are means ± SEM. Data were analyzed using a two-tailed non-paired students t-test (e) as well as a two-way repeated ANOVA test (one factor repeated) and two-tailed Student-Newman-Keuls post hoc analyses for multiple comparisons (b, c). AU, arbitrary units. Scale bar in panel e is 10 µm. Source data

Extended Data Fig. 4. Insulin- and AMPK-related signaling is not compromised in skeletal muscle from homozygous TBC1D4 p.Arg684Ter variant carriers after a single bout of exercise.

a–g, Skeletal muscle protein phosphorylation of Akt Ser473 (a), GS site 2 + 2a (Ser7 + Ser10) (b), GS site 3a + 3b (Ser640 + Ser644) (c), TBC1D4 Ser318 (d), TBC1D4 Ser341 (e), TBC1D4 Ser588 (f) and AMPKα Thr172 (g) in rested and prior exercised muscle before and at the end of the insulin clamp. h, Representative immunoblots. i, The top 15 up- and downregulated Gene Ontology (GO) pathways of the proteome. n = 5 (Rested and Exercised leg – insulin), n = 3 (Rested leg + insulin), and n = 4 (Exercised leg + insulin) in Controls and n = 5 in TBC1D4 carriers (a–g). The difference in n is due to missing biopsies as well as depleted sample material. n = 5 (i). Data are means ± SEM. Data were analyzed using a two-way repeated ANOVA test (two factor repeated) and two-tailed Student-Newman-Keuls post hoc analyses for multiple comparisons (a–g). AU, arbitrary units. LLM, leg lean mass. Source data

Extended Data Fig. 5. Leg glucose uptake, blood flow and arterial-venous glucose difference during euglycemic-hyperinsulinemic clamp conditions as well as whole-body substrate utilization and mitochondrial respiration in permeabilized muscle fibers from homozygous TBC1D4 p.Arg684Ter variant carriers.

a, b, Respiratory Exchange Ratio (RER) during an extended (6-h) oral glucose tolerance test (OGTT) (a) as well as before and at the end of the insulin clamp (b). c, Mitochondrial respiration rate (O₂ flux) in permeabilized muscle fibers in the presence or absence of acylcarnitines (palmitoylcarnitine/octanoylcarnitine). d–f, Glucose uptake (d), blood flow (e) and arterial-venous glucose difference (f) in the prior rested and exercised leg during the 120 min insulin clamp. ‡Different from fasting in both groups. n = 5 in both groups (b–e) and n = 8 in Controls and n = 7 in TBC1D4 carriers (a). Data are means ± SEM. Data were analyzed using a two-way repeated ANOVA test (one factor repeated (a) and two factor repeated (b–e)) and two-tailed Student-Newman-Keuls post hoc analyses for multiple comparisons (a–e). LLM, leg lean mass.

Extended Data Fig. 6. Leg blood flow, leg arterial-venous glucose difference, blood glucose, plasma hormones and skeletal muscle genes are regulated similarly in Controls and homozygous TBC1D4 p.Arg684Ter variant carriers during and in recovery from exercise.

a, b, Leg blood flow (a) and leg arterial-venous glucose difference during 1 h of knee-extensor exercise and 3 h recovery. c–h, Concentrations of blood glucose (c), plasma adrenaline (d), plasma noradrenaline (e), blood lactate (f), plasma insulin (g) and plasma fatty acids (h) before, during and 3 h into recovery from exercise. i, j, Skeletal muscle protein phosphorylation of glycogen synthase (GS) site 3a + 3b (i) and pyruvate dehydrogenase (PDH) site 2 (Ser300) (j) in the previously rested leg (Rest) as well as immediately after exercise in the exercised leg (Exercise). Representative immunoblots are shown below the data panels. k, Regulation of selected skeletal muscle genes 3 h into recovery from exercise in Controls and TBC1D4 carriers. The selected genes have previously been reported top exercise responsive with acute aerobic exercise (see ref. in main article). The selected genes are indicated by “Gene”, while the Log2 Fold Changes of the exercise response between Controls and TBC1D4 carriers are indicated by “Log2 FC”. Positive values indicate a higher exercise response in TBC1D4 carriers than in Controls, while negative values indicate a lower exercise response in TBC1D4 carriers than in Controls. The nominal p values are indicated by “p value”, while the adjusted p values used to correct for multiple testing are indicated by “p adj”. ‡Different from Rest (time=0) within both groups. n = 5 in Controls and in TBC1D4 carriers. Data are means ± SEM. Data were analyzed using a two-way repeated ANOVA test (two factor repeated: time/group and genotype) and two-tailed Student-Newman-Keuls post hoc analyses for multiple comparisons. AU, arbitrary units. Source data